The purpose of this proposal is to foster the clinical investigation and laboratory research skills of Dr. Lei Zheng as he transitions into an independent translational researcher with clinical expertise in GI cancers, and research expertise in pancreatic cancer immunotherapy and the clinical development of new therapies that target the tumor's microenvironment. Dr. Elizabeth Jaffee will serve as Dr. Zheng's primary mentor. The Sidney Kimmel Cancer Center at Johns Hopkins and its Immunology and Gastrointestinal Cancer Programs provide a unique training environment to develop novel immune based combinatorial therapy approaches for the treatment of pancreatic ductal adenocarcinoma (PDA). As an initial translational research study, this proposal will investigate the association between inhibition of T regulatory cells (Tregs), parameters of immune response, and clinical responses, in patients with resectable PDA treated in the neoadjuvant and adjuvant settings with a GM-CSF secreting allogeneic pancreatic cancer cell vaccine. This vaccine alone will be compared with vaccine given in sequence with either a single intravenous dose or daily metronomic oral doses of cyclophosphamide (Cy) as approaches for inhibiting Tregs. This study also provides the unique opportunity to compare the systemic induction of T cell responses with responses at the tumor site following therapy. Previously, Dr. Jaffee's group has shown that this vaccine is safe and enhances survival in phase I and II studies. The survival benefit also correlates with the induction of T cells specific for mesothelin, a candidate PDA antigen. However, immune tolerance remains the major obstacle to effective immune- based treatment in cancer patients. Tregs in particular, reside at the tumor site and inhibit the function of effector T cells. A single intravenous dose of Cy has been shown to transiently deplete Tregs in pre- and early clinical studies. Repetitive doses of metronomic Cy were shown to lead to long-term and selective inhibition of Tregs. This project will assess the effects of Cy depletion on the tumor-infiltrating lymphocyte population, on vaccine-induced tertiary lymphoid structures, and on the expression of two novel immune regulatory molecules, Annexin A2 and galectin-3, that were recently shown to have altered expression in PDA. We will recruit 39 evaluable patients with resectable, suspected PDA into a three-arm pilot study and assess the safety, feasibility, and immune responses associated with an allogeneic GM-CSF-secreting pancreatic tumor vaccine administered either alone or in combination with a single intravenous dose or daily metronomic oral doses of Cy as neoadjuvant and adjuvant treatment. In vivo parameters of immune responses will be compared in patients treated in each arm. This study will test the hypothesis that treatment with metronomic Cy more effectively depletes or inhibits Treg function within the primary tumor, and results in the induction of a higher avidity and a more diverse mesothelin-specific T cell repertoire. Finally, this study allows, for the first time, a rigorous analysis of the effects of combinatorial immunotherapy on the composition of lymphocytes infiltrating the tumor's microenvironment. This study provides the opportunity to explore the interactions of Tregs and two proteins, Annexin A2 and galectin-3, that have been linked to immune suppression and the progression of PDA.